Carvalho et al. studied the importance of the eutopic endometrium in the development of pelvic endometriosis [29]. Although the morphology of the cells in eutopic endometrium of women with and without the disease is similar, there are some differences in the biochemistry, function and genetics among these cells. These differences may be one of the factors contributing to the development of endometriosis.

Evidence suggests that the density of nervous fibers in women with endometriosis is increased, although it is not known if this is correlated with the disease or with pelvic pain [18]. Thereby, some neural transmitters appear to be increased in endometriotic women. NT-4/5 and brain-derived neurotrophic factor proteins were found to be over-expressed in women with endometriosis [34]. PGP9.5 immuno-active nerve fibers were suggested to predict endometriosis with sensitivity of 98 % and specificity of 83 % [35]. A combination of PGP9.5, vasoactive intestinal peptide and substance P was also studied and was shown to have a 95 % sensitivity and 100 % specificity [36].

VEGF was also found to be up-regulated in eutopic endometrial tissue, mainly in the late secretory phase and during menstruation of women with endometriosis [37] (Fig. 9.2).

Ren et al. studied the effect of ischemic precondition (IPC) in endometriotic women. The researchers hypothesized that the endometrium becomes slightly ischemic during the early and middle secretory phase, mimicking an IPC response. They also found that this IPC lead to an increase in VEGF expression and a decrease in apoptosis, therefore facilitating angiogenesis and implantation of endometrial cells [38]. Additionally, there seems to be a relationship between oxidative stress and VEGF. Schafer et al. showed that an increase in ROS levels leads to an increase in VEGF levels [39].

Annexin V plays a role in proliferation and cell mobility. It was up-regulated in the eutopic endometrium of women with endometriosis, showing a possible relation to the implantation of endometriotic tissue. T plastin plays a role in cell locomotion and maintenance of cellular architecture. It was reported to be up-regulated in the eutopic endometrium of women with endometriosis [36]. Further investigation about its correlation with the pathophysiology of the disease is needed, but it may play also play a role in implantation of endometrial cells outside the uterine cavity. Both of these proteins were studied using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and their increase show they might influence the migration of endometrial cells outside the uterine cavity.

Caldesmon is a protein that binds actin, inhibiting ATPase activity. In non-muscle cells, it inhibits motility and phagocytosis. Meola et al. studied the expression of the CALD1 gene, which encodes caldesmon, and the levels of caldesmon in eutopic and ectopic endometrium. Real-time PCR and Western blot analysis and immunostaining to determine cellular localization were used. No variation among the cells was found in the immunostaining. Caldesmon levels were found to be lower in the eutopic endometrium of women with endometriosis [40]. The study included patients with other gynecologic diseases, which is a large limitation. However, the findings showed that this protein may play a role in the disease. Once it is diminished, it cannot bind to actin and, thus, is unable to inhibit cell motility.

Stephens et al. used a proteomic approach to study differently expressed proteins in eutopic endometrium [17]. Glucocorticoid receptor subunit alpha (GCR), a protein that can bind progesterone, was found to be highly expressed among endometriotic women. Because endometriosis is associated with progesterone resistance, it may play a role in the pathophysiology of the disease.

Heat shock protein (HSP) is a chaperone protein that accumulates in cells under stressful conditions. It plays a role in (un)folding and transporting proteins, controlling the cellular cycle, counteracting the effect of oxidative stress and modulating apoptosis [8]. It was found to be increased in endometriosis, suggesting an increased oxidative stress in endometrial cells of women with endometriosis.

Superoxide dismutase (SOD), an enzymatic anti-oxidant, and peroxide, which forms as a result of oxidative stress, were both up-regulated in eutopic endometrium. SOD may be high because it is compensating for the OS [41].

Thioredoxin is an anti-oxidant protein and is involved in apoptosis and cellular proliferation. Thioredoxin binding protein 2 (TBP-2) regulates thioredoxin and promotes apoptosis when a cell has a high level of oxidative stress. There were no significant differences in the levels of TRX or TBP in endometriosis. However, the ratio of TRX to TBP and TRX/TBP was increased in endometriotic lesions, As a result, the anti-oxidant capacity is decreased while cell proliferation is increased, [42] suggesting that these proteins may be related to the development of endometriosis. The methodology of the study included real-time PCR and immunohistochemistry in endometrial tissue.

IL1 soluble receptor accessory protein, (s)IL1RAcP, inhibits secretion of IL1. It was significantly down-regulated in eutopic endometrium of women with endometriosis, at the mRNA and the protein levels in the secretory phase and in glandular and surface epithelial cells. The membrane-bound IL1 receptor accessory protein, (mb)IL1RAcP, was not correlated with the presence of the disease, neither at the mRNA nor the protein levels [43]. IL1 decoy inhibitory receptor, IL1R2, was significantly decreased in eutopic endometrium of women with endometriosis. These associated findings show that there is an imbalance in IL-1 production. As IL-1 is associated with the capacity of endometrium implantation, these results are in accordance with the pathophysiology of the disease [44].

Some apoptotic molecules were shown to be differently expressed. Bcl-2, an anti-apoptotic protein, is over expressed in stromal cells in proliferative eutopic endometrium. Additionally, Bax, a pro-apoptotic protein, was absent in proliferative endometrium and increased in the endometrium of endometriotic women [45].

In the eutopic endometrium, a large amount of proteins reflecting various processes were identified. Alterations in the immune system, oxidative stress and inflammation markers along with high levels of cell migration, motility, proliferation and adhesion were once again observed. However, in this biological window, there were some new findings. A progesterone resistance was identified. Furthermore, a higher density of nervous fibers was found, and some neural transmitters were found to be highly expressed. Nonetheless, this finding needs further investigation in order to elucidate whether the nerve fibers are related to the disease or to pelvic pain. It may be a factor that contributes to the chronic pain seen in endometriotic women.

Follicular fluid contains secretions from the ovarian follicles and is an ultra-filtrate from the blood plasma. It contains many elements such as proteins, hormones and enzymes and therefore, it is a reliable biological fluid that can be studied for biomarkers and to discover the underlying causes of the disease. Lo Turco et al. [46] compared the follicular fluid between three groups of women; healthy controls (C), endometriotic women who achieved pregnancy (E.P) and endometriotic who did not achieve pregnancy (E.NP) Proteins were separated by 2DE and compared and identified by LC-ESI-MS-MS (Refer Fig. 9.4). Serum albumin was significantly down-regulated in the E.P and E.PN groups. It is a protein whose functions are binding to DNA, copper and fatty acids. It also has antioxidant activity. Therefore, low levels may indicate the presence of oxidative stress. This finding is in accordance to the oxidative stress pathophysiology of endometriosis.

Complement Factor I is typically a serum protein and also a glioma and lung-cancer protein. It was first found in follicular fluid and down regulates complement activation. The complement system is composed of proteins that, when activated, kill invasive pathogens and destroy non-self-molecules (Fig. 9.4). Complement Factor H, which has similar functions, was up-regulated in the E.P group. This highlights the importance of an altered immune system in the pathogenesis of endometriosis. Angiotensinogen, a growth factor, was found to be highly expressed in the E.P and E.NP groups, showing that this protein may contribute to the proliferation of ectopic tissue. Vitronectin, an integrin-binding protein and also a component of the extracellular matrix protein, was found to be overexpressed in the E.NP group [46]. It may play a role in the adhesion process of endometriosis (Fig. 9.4). Focal adhesion kinase 1 is a protein found in adhesion sites of cells and is associated with cell migration and survival [47]. It was highly expressed in the E.P group, showing that it may play a role in the development and progression of the disease, as adhesion is one of the main characteristics of endometriosis. Kininogen-1 protein, which participates in blood coagulation, was found to be increased only in the E.NP group [46]. Jarkovska et al. showed that there is a link between Kininogen-1 protein and VEGF. Therefore, this increase may contribute to adhesion and neovascularization—two common processes in endometriosis [48].

Fas antigen in NK-cells were found to be highly expressed in the peritoneal fluid of women with endometriosis. It suggests that the elimination of NK cells provides allows ectopic endometrium to survive. This finding underlies the importance of a deficient immune system in the development of endometriosis [49].

Prieto et al. compared levels of OS markers among infertile women with endometriosis and infertile women due to other conditions. Vitamin C, a non-enzymatic anti-oxidant, was decreased in the follicular fluid of women with endometriosis. Superoxide dismutase, on the other hand, was decreased in the plasma of endometriotic women [20].

Investigating the protein expression of ectopic endometrial tissue may lead to the discovery of some biomarkers and, also, lead to a better understanding of the pathophysiology of this enigmatic disease.

The CALD1 gene, which encodes the protein caldesmon, was analyzed as a potential biomarker to diagnose endometriosis (Fig. 9.2). Endometrial tissue from eutopic and ectopic endometrium was obtained from women with endometriosis and levels of CALD1 gene and caldesmon protein were determined by PCR, western blot and immunostaining. The results found that they were increased in the endometriotic tissue. It was found that the protein caldesmon can predict endometrial dysregulation in women with endometriosis [40].